SUMMARY Calorie restriction (CR), or enforced reduced food intake without malnutrition, is highly beneficial on glucose and lipid homeostasis, acute stress resistance and longevity in multiple experimental organisms. Restriction of dietary sulfur amino acids methionine and cysteine, known as methionine restriction (MR), also results in most of the same benefits in experimental rodents, but without enforced calorie restriction, and thus potentially by a different underlying mechanism. In the prior funding period we discovered a novel molecular mechanism underlying pleiotropic benefits common to both CR and MR: increased production of endogenous hydrogen sulfide gas (H2S) via the evolutionarily conserved transsulfuration pathway (TSP). In particular, we found that restriction specifically of cysteine intake increased expression of the TSP enzyme cystathionine ?-lyase (CGL), resulting in increased H2S. The health benefits of H2S have only recently begun to be appreciated. Engineered CGL knockout mice lacking the ability to produce adequate endogenous H2S have high blood pressure and susceptibility to neurodegenerative disease, while exogenous H2S addition can act as a vasodilator, immunomodulator, and neuroprotectant in experimental rodents, and even increase lifespan in lower organisms. Our finding that increased endogenous H2S is necessary and sufficient for two of the major health benefits of CR, namely increased stress resistance and extended longevity across evolutionary boundaries, cements the notion that increased endogenous H2S is highly beneficial to numerous health outcomes. Our findings also justify the major goal of this proposal, namely to understand novel interventional approaches to increasing endogenous H2S production, and applying of these findings to areas of high clinical relevance. Here we propose to test the hypothesis that diets low in protein, and particular low in the sulfur amino acids methionine and cysteine as found in vegetable-derived proteins, result in increased CGL expression and H2S production in part through activation of the amino acid deprivation sensor GCN2. We will also test the hypothesis that the substrate for endogenous H2S production is derived from autophagy, linking this effector of longevity benefits to H2S production for the first time. Finally, we will explore the novel role of H2S in protection of hematopoietic stem cells against ionizing radiation, and test a novel mechanism of increasing H2S delivery to tissues such as bone marrow involving endocrine action of circulating CGL protein.